28/05/2014 THE HOST: RESISTANCE GENE ISOLATION AND REALISING THE POTENTIAL PLANT PATHOGENS ARE PICKY

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1 THE HOST: RESISTANCE GENE ISOLATION AND REALISING THE POTENTIAL Kim Hammond-Kosack Wheat Pathogenesis Programme Rothamsted Research PLANT PATHOGENS ARE PICKY Most plants are naturally resistant to most pathogenic microbial species Plant disease is rare 19 th December 2006 ESCAPE R PROTEIN RACE - SPECIFIC RESISTANCE TOLERANCE PREFORMED STRUCTURAL BARRIERS and ANTIMICROBIAL COMPOUNDS NON-HOST RESISTANCE BASAL RESISTANCE RACE - SPECIFIC GENETIC INTERACTIONS FLOR s Gene-for-Gene Hypothesis Pathogen genotype Avr1, avr2 avr1, Avr2 Host plant genotype R1 r2 I C r1 R2 C I - incompatible - no disease C - compatible - disease I 3 COMPONENTS TO INDUCIBLE DEFENCE RESISTANCE GENE ISOLATION PATHOGEN RECOGNITION DEFENCE SIGNALLING R PROTEINS ONLY FUNCTION HERE Started in late 1980s Several groups successful by 1994 Approach molecular genetics A MULTI-COMPONENT RESISTANCE RESPONSE 1

2 MAP - BASED ISOLATION OF R GENES MAP - BASED ISOLATION OF R GENES Genetic marker 1 R gene Genetic marker 2 M3 M4 5. Transform a susceptible genotype with a single cosmid clone Disease reaction R R S S Types of problems encountered - A LONGER WALK than expected - lack of recombination - An unknown HOLE IN THE BAC CLONE RPM1 - Which gene is it? PTO TRANSPOSON TAGGING OF R GENES The two-component system TRANSPOSON TAGGING OF R GENES with genetic selection Tobacco N gene Tomato Cf-9 : Avr9 30 C 22 C TMV Ac transposon in a T-DNA R gene Ds transposase gene in a T-DNA nn Rare outcome inactive R gene or NN Transposon inactivation of R gene RESISTANCE GENES ISOLATED Host species Model Crop Monocotyledonous Dicotyledonous Effective against Fungi Oomycetes Bacteria Viruses Nematodes Aphids Whiteflies What protein types do R genes code for? 2

3 RACE SPECIFIC R PROTEINS Xa21 Pi-d2 RACE NON-SPECIFIC R PROTEINS RACE SPECIFIC R PROTEIN (SUMMARY) cytoplasm NLS WRKY RRS1 N, L6 RPP5 Cf-2, 4, 5, 9 RPS2 RPM1 1 BS2 Ve1 Ve2 PEST ECS kinase Pto PBS1 5 6 kinase kinase Xa27 2 RPW8 3 4 RPG1 kinase kinase Still a very limited number of R protein types and motifs A single R protein class can confer resistance to different pathogen types Intracellular -- bacteria, fungi (Basidiomycetes and Ascomycetes), Oomycetes, virus, root knot and cyst nematodes and an aphid A single pathogen species can be controlled by different types of R protein Hyaloperonospora parasitica Downy mildew of Arabidopsis e RPP8 gene RRP4 gene RPP27 gene* * Retracted by authors Jan 2007 Plant Physiology see original article R PROTEIN / R LOCI SPECIES SPECIFICITY R gene expression Most R proteins / R loci confer resistance to just specific isolates within a single species A few exceptions Tomato Mi protein ( --) - root knot nematode, aphid and whitefly Potato - neighbouring genes Rx1 and Gpa2 Both -- types (88% amino acid identity) Rx1 Gpa2 potato virus X cyst nematode Very low and constitutive Occasionally low level induction following infection but only in the vicinity of the pathogen Rarely expressed only in resistant genotype - Rice Xa27 Xanthomonas oryzae pv. oryzae Susceptible alleles 10 bp and a 25 bp insertion in the promoter (Gu et al, (2005) Science 435: ) How do R proteins function? Four examples Direct pathogen molecule recognition Indirect pathogen molecule recognition Susceptible Direct recognition Tomato Pto - AvrPto Pseudomonas syringae pto AvrPto and AvrPtoB effectors bind to unknown target Enhanced pathogen virulence Adapted from Jones and Dangl (2006) Nature AvrPto or AvrPtoB Pto Prf HR Less pathogen proliferation Resistant Pto plasma membrane 3

4 Indirect recognition Arabidopsis RPM1- AvrRpm1 R PROTEINS GUARD THE VIRULENCE TARGET Susceptible NDR1 AvrRpm1 or AvrB Pseudomonas syringae NDR1 Resistant RPM1 A. COMPATIBLE INTERACTION Avr Virulence Target Susceptible response to favour pathogen growth and development No RPM1 RIN4 P P P RIN4 P P P B. INCOMPATIBLE INTERACTION Resistance by guarding AvrRpm1 effector bind to target RIN4 and other targets RPM1 Avr Virulence Target R3 Resistance response Enhanced pathogen virulence Adapted for Jones and Dangl (2006) Nature HR Less pathogen proliferation AvrB/ RIN4 RPM1 AvrRpm1 Avr2 Rcr3 Cf-2 Tomato Cf-4 Cladosporium fulvum Avr4 Barley Mla10 and powdey mildew Avra10 Susceptible Chitinase Avr4 Enhanced pathogen virulence Fungal chitin Suzan Gabriels, Matthieu Joosten (2007) Plant J (in press) Avr4 Cf-4 Rcr3 Avr4 Activation of Cf dependent defence response Less pathogen proliferation Cf-4 e Resistant NRC1 -- Susceptible appressorium penetration peg Modified plant Avra10 effector bind to target Enhanced pathogen virulence haustorium Avra10 EHM fungal plasma membrane plant cell cytoplasm ECM extracellular haustorial matrix or spore? Plant? nucleus Shen, Q-H Schultz-Lefert (Science 21 st Dec 2006) Chris Ridout (2006) Plant Cell 18, WRKY1/2 Resistant Mla10 HR Less pathogen proliferation Pto kinase R PROTEIN DEFENCE ACTIVATION Cytoplasmic route -- NDR1 MAP kinase cascades RAR1 SGT1 HSP90 HR NO -- OB early EDS1 SA NPR1 RPW8 late Early cytoplasmic to nuclear shuttling of R proteins Plant nucleus ---NLS-WRKY TGAs -- WRKY W box STUBBORNLY UNANSWERED QUESTIONS 1. How to extracellular -TM class of R proteins work? Cladosporium fulvum Small secreted Avr peptides Avr9 Avr4 Avr2 Avr5 Tomato Cf-2, Cf-4 Cf-5, Cf-9 N-terminal confer specificity (24 aa) Early defence responses PR and other defence proteins cytoplasm 4

5 STUBBORNLY UNANSWERED QUESTIONS STUBBORNLY UNANSWERED QUESTIONS (2) 2. What is the crystal structure of a plant domain? 3. What other types of targets are guarded by R proteins? 4. What function(s) to these targets provide to successful pathogens? 5. Why do only a few R genes confer durable resistance? Tomato Tm2 2, Barley Rpg1 Porcine ribonuclease inhibitor < 30% amino acid identity with plant containing proteins NEW INFORMATION OBTAINED THROUGH SEQUENCING PLANT GENOMES THE POST - GENOMICS ERA What are the possibilities? R protein class RLP (e-tm and ellr-tm-kinase) Putative R gene numbers Arabidopsis Rice Poplar Proteomics phospho-proteomics Use of epitope tagged R proteins to identify the direct interactors FRET Fluorescence resonance energy transfer to confirm interactions in vivo 2. VIGS Virus induced gene silencing to explore gene function in a high throughput mode -- common ancestral origin Some gene classes have other functions plant development ß Identification of wheat leaf rust resistance gene Lr21 VIGS vector a a ßb ßd ßc PacI a b plant seq nt Barley stripe mosaic virus NotI Scofield et al. (2005) Plant Physiology 138: S R A B R GENE APPLICATIONS REALISING THE POTENTIAL Non- transgenic approaches 1. Development of markers within-the-r gene or closely linked to the R gene Barley rym4 soil-borne viruses - Useful in R gene introgression by plant breeders - minimises the need for bioassays R R R S S R S R R R 2. Pyramiding of R genes each working in a different way is a real possibility K Kanyuka 5

6 EACH RACE SPECIFIC R GENES HAS ONLY A LIMITED LIFE SPAN BOOM YEARS R GENE APPLICATIONS REALISING THE POTENTIAL Transgenic approaches Sophisticated Simple BUST YEARS THE CONSEQUENCES OF CONSTITUTIVELY ACTIVATING PLANT DEFENCES PATHOGEN - INDUCIBLE PROMOTERS Plant death Reduced plant growth rates 30 C 22 C TMV Tomato Cf-9 : Avr9 Slower plant development nn Lower crop yield NN Transposon inactivation of R gene Synthetic promoters pathogen inducible Durable disease control achieved by using two contrasting transgenes in each T-DNA cassette add motif tetramers from WRKY promoter Hyaloperonospora parasitica Arabidopsis CaMV 35S 4 x W1 GUS LB Plant defence ppi Gene 1 Examples Self activated R* protein Broader recognition R protein Pathogen debilitation pp2 Gene 2 Variant virulence target RB Shuffled chitinase/glucanase Incompatible Improved defence signalling protein Enzyme to block / degrade a pathogenicity factor Paul Rushton et al. (2002) The Plant Cell 14: Major QTL enzyme P - borders 6

7 R GENE APPLICATIONS REALISING THE POTENTIAL UK GM field trials for 2007 Transgenic approaches Announced Dec 2006 by BASF Control of late blight by two R genes from potato Sophisticated Simple Rpi-blb1 and Rpi-blb2 -- proteins evolutionarily related to tomato Mi Cloning of Rpi-blb2 van der Voissen et al., (2005) Plant Journal 44, UK GM late blight field trials for 2007 SUMMARY (1) Rpi-blb1 and Rpi-blb2 both originally from the diploid potato species Solanum bulbocastanum Both confer high level resistance to P. infestans isolates with complex race structures BROAD SPECTRUM Transfer of resistance Tedious and time-consuming breeding schemes involving ploidy manipulations and a series of bridge crosses gave P. infestans resistant inter specific hybrids - ABPT lines ABPT lines in field trials at multiple sites in Netherlands over 20 years gave no or only a few sporulating lesions at the end of the season Organic and intensive farming conditions tested Isolation of the first R genes was difficult now far easier through homology searches and transient transformation techniques R proteins possess both recognition and signalling domains Limited number of R protein classes with conserved structural domains -s - largest class no -- in monocots Pathogen effector (Avr) signals are far more variable SUMMARY (2) Many thanks to Some R proteins may guard pathogen virulence targets Plant R proteins function only in closely related species Several different types of R locus organisation Many unanswered questions on R protein function as well as R gene evolution Exploitation - Marker assisted selection and breeding - Transformation of broad spectrum and durable R genes into elite genotypes - Sophisticated R-Avr cassettes and R* genes using pathogen inducible promoters Rothamsted Research Kostya Kanyuka Hai-Chun Jing Jason Rudd John Lucas Ian Crute Elsewhere Jonathan Jones Matthieu Joosten Paul Schultz-Lefert Chris Ridout Greg Martin 7

8 :: - RACE-SPECIFIC R PROTEINs Extra slides Plants N - tobacco L6 - flax RPP5 - Arabidopsis Drosophila Toll Mammals Interleukin receptors extracellular cytoplasm Dorsal - ventral polarity and defence Immunity and defence :: - RACE-SPECIFIC R PROTEINs N - tobacco L6 - flax RPP5 - Arabidopsis cytoplasm Plants TLRs Mammals Signals - LPS, unmethylated DNA flagellin extracellular Immunity and CARD defence NOD 1, NOD 2 THE LEUCINE-RICH REPEAT DOMAIN Contains the reiterated sequence missing LxxLxxLxxLLxLxx(N)xLxGxIPxx b - strand / b - turn structural motif 23 aa (I) 24 aa (E) Within this motif, leucine residues project into the hydrophobic R protein core whilst x amino acids are solvent exposed and very variable Detection of pathogen - derived signals? TRANSCRIPTION PROFILING TO IDENTIFY PLANT PROMOTERS INDUCED BY MULTIPLE PATHOGENS Control healthy infected RNA Probe sources Different biotrophic pathogens Different necrotrophic pathogens overlay images and normalise Compatible interactions analysis BIOINFORMATICs (eg. MEME) to identify common motifs in promoters (arabidopsis, rice) 8